1. Technical Field
The invention relates to methods and materials involved in the transformation of Brassica by particle bombardment.
2. Background Information
Brassica species include a large group of agriculturally important crops that are used by humans as vegetables, edible oils, and condiments. In fact, Brassica oil production accounts for more than 12 percent of the world""s edible oil. To improve the quality of agriculturally important crops, cultivators have traditionally relied upon conventional breeding methods. With the current advances in plant molecular biology and genetics, however, cultivators can now improve plant quality through the introduction of foreign DNA.
Several different methods have been used to transform plants. One commonly used method involves bombarding plant cells with microparticles that have been coated with the DNA of interest. Indeed, particle bombardment methods have been widely used to transform corn, soybean, wheat, and rice. Attempts to transform Brassica species using particle bombardment, however, have not been as successful. In fact, the only successful transformation of Brassica required substantial manipulation of Brassica embryos. Thus, researchers currently rely on alternative approaches such as Agrobacterium-mediated methods to transform Brassica.
The invention involves the transformation of Brassica species by particle bombardment. Specifically, the invention is based on the discovery of a quick and convenient tissue preparation technique that results in Brassica cells that are capable of being cultured, transformed by particle bombardment, and regenerated into plants. In addition, the invention provides transformed cells as well as regenerated plants that grow to maturity and set seeds. Such regenerated plants and their offspring stably express the transferred nucleic acid molecule. The simplicity of the invented method described herein makes particle bombardment transformation in Brassica not only possible, but also economically feasible.
One aspect of the invention provides a method of producing transformed Brassica cells involving bombarding cells prepared from non-embryo Brassica tissue with nucleic acid-coated microprojectiles and identifying cells transformed with the nucleic acid. Cells transformed with the nucleic acid can be identified by placing the bombarded cells onto a floatation device, contacting the floatation device with liquid selection medium, and selecting cells that survive. This method can also involve regenerating Brassica plants from the transformed cells. For example, identified cells can be placed onto a floatation device that contacts liquid regeneration medium. The cells prepared from non-embryo Brassica tissue can be diploid. In addition, the non-embryo Brassica tissue can be from a seedling, for example, a 4-6 day old sterile seedling. The cells can be prepared from non-embryo Brassica tissue by cutting the tissue into pieces and contacting the pieces with induction medium. For example, the cells can be prepared from non-embryo Brassica tissue by a method comprising cutting a hypocotyl from a seedling into multiple pieces, slicing the pieces longitudinally, and contacting the epidermal side of the longitudinal slices with induction medium. The cells can also be prepared from non-embryo Brassica tissue by a method comprising macerating the tissue into a cellular slurry. For example, the cells can be prepared from non-embryo Brassica tissue by a method comprising removing the lower portion of a hypocotyl from a seedling, combining the remaining upper portion, which contains a shoot tip, cotyledons, and 10-50 percent of the hypocotyl, with induction medium, and macerating the combination into a cellular slurry. In addition, the cellular slurry can also be enriched for 46-230 micron-sized cellular matter. The nucleic acid used to transform Brassica cells can regulate the expression of or encode a polypeptide. For example, the nucleic acid can encode an anti-sense molecule, such as a ribozyme or anti-sense oligonucleotide. Further, the Brassica tissue can be from Brassica species such as Brassica napus, Brassica juncea, Brassica carinata, Brassica nigra, Brassica oleracea, and Brassica campestris (also called xe2x80x9crapaxe2x80x9d).
Another aspect of the invention provides Brassica cells and progeny thereof produced by bombarding cells prepared from non-embryo Brassica tissue with nucleic acid-coated microprojectiles and identifying cells transformed with the nucleic acid.
Another aspect of the invention provides a method for culturing Brassica tissue involving placing Brassica tissue onto a floatation device that contacts liquid medium. This liquid medium can be selection medium. Culturing Brassica tissue on selection medium can result in the identification of Brassica tissue that has been transformed with nucleic acid. In addition, this liquid medium can be regeneration medium. Culturing Brassica tissue on regeneration medium can result in the regeneration of a Brassica plant from Brassica tissue.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.